1. Field of the Invention
This invention relates to detection of mutated nucleic acid and genetic polymorphisms by single base extension analysis. Specifically, the invention relates to single base extension following hybridization of a biological sample comprising a nucleic acid with an oligonucleotide array. In particular, the invention provides apparatus and methods for electronic detection of single base extension of a particular oligonucleotide in an oligonucleotide array after hybridization to a nucleic acid in a biological sample and single base extension thereof.
2. Background of the Invention
The detection of single base mutations and genetic polymorphisms in nucleic acids is an important tool in modern diagnostic medicine and biological research. In addition, nucleic acid-based assays also play an important role in identifying infectious microorganisms such as bacteria and viruses, in assessing levels of both normal and defective gene expression, and in detecting and identifying mutant genes associated with disease such as oncogenes. Improvements in the speed, efficiency, economy and specificity of such assays are thus significant needs in the medical arts.
Ideally, such assays should be sensitive, specific and easily amenable to automation. Efforts to improve sensitivity in nucleic acid assays are known in the prior art. For example, the polymerase chain reaction (Mullis, U.S. Pat. No. 4,683,195, issued Jul. 28, 1987) provides the capacity to produce useful amounts (about 1 xcexcg) of a specific nucleic acid in a sample in which the original amount of the specific nucleic acid is substantially smaller (about 1 pg). However, the prior art has been much less successful in improving specificity of nucleic acid hybridization assays.
The specificity of nucleic acid assays is determined by the extent of molecular complementary of hybridization between probe and target sequences. Although it is theoretically possible to distinguish complementary targets from one or two mismatched targets under rigorously-defined conditions, the dependence of hybridization on target/probe concentration and hybridization conditions limits the extent to which hybridization mismatch can be used to reliably detect, inter alias, mutations and genetic polymorphisms.
Detection of single base extension has been used for mutation and genetic polymorphism detection in the prior art.
U.S. Pat. No. 5,925,520 disclosed a method for detecting genetic polymorphisms using single base extension and capture groups on oligonucleotide probes using at least two types of di deoxy, chain-terminating nucleotide triphosphate, each labeled with a detectable and distinguishable fluorescent labeling group.
U.S. Pat. No. 5,710,028 disclosed a method of determining the identity of nucleotide bases at specific positions in nucleic acids of interest, using detectably-labeled chain-terminating nucleotides, each detectably and distinguishably labeled with a fluorescent labeling group.
U.S. Pat. No. 5,547,839 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, using chain-terminating nucleotides comprising a photo removable protecting group.
U.S. Pat. No. 5,534,424 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, using each of four aliquots of a target nucleic acid annealed to an extension primer and extended with one of four chain-terminating species, and then further extended with all four chain-extending nucleotides, whereby the identity of the nucleotide at the position of interest is identified by failure of the primer to be extended more that a single base.
U.S. Pat. No. 4,988,617 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, by annealing two adjacent nucleotide primers to a target nucleic acid and providing a linking agent such as a ligase that covalently links the two oligonucleotides to produce a third, combined oligonucleotide only under circumstances wherein the two oligonucleotides are perfectly matched to the target nucleic acid at the 3xe2x80x2 extent of the first oligonucleotide and at the 5xe2x80x2 extent of the second oligonucleotide.
U.S. Pat. No. 4,656,127 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, using primer extension with a chain-terminating or other nucleotide comprising an exonuclease-resistant linkage, followed by exonuclease treatment of the plurality of extension products to detect the resistant species therein. One common feature in this prior art is that single base extension has been detected by incorporation of fluorescent labels into the extended nucleic acid species.
A significant drawback of single base extension methods based on fluorescent label detection is the need for expensive and technically-complex optical components for detecting the fluorescent label. Although fluorescent probes used in such methods impart an adequate level of discrimination between extended and unextended positions in an oligonucleotide array, these methods typically require detection of up to four different fluorescent labels, each having a unique excitation and fluorescence emission frequency. As a consequence of these properties, such assay systems must be capable of producing and distinguishing light at all of these different excitation and emission frequencies, significantly increasing the cost and complexity of producing and operating apparatus used in the practice thereof.
An alternative method for detecting a target nucleic acid molecule is to use an electrochemical tag (or label) such as a redox moiety in combination with an electrochemical detection means such as cyclic voltammetry.
U.S. Pat. No. 5,591,578 provides for the selective covalent modification of nucleic acids with redox-active moieties such as transition metal complexes of specifically-claimed transition metals, wherein the complexes are covalently linked to a ribose sugar comprising the ribose-phosphate backbone. The resulting complexes are capable of transferring electrons over very large distances at extremely fast rates.
U.S. Pat. No. 5,705,348, related to U.S. Pat. No. 5,591,578, encompasses generally selective covalent modification of nucleic acids with redox-active moieties such as transition metal complexes, wherein the transition metals are generically-claimed.
U.S. Pat. No. 5,770,369, related to U.S. Pat. No. 5,591,578 discloses electron donor and acceptor moieties that are not redox proteins.
U.S. Pat. No. 5,780,234 369, related to U.S. Pat. No. 5,591,578, discloses methods wherein two single stranded nucleic acid are used to hybridize to two different domains of the target sequence.
In addition, disclosure of similar methods for detecting biological molecules such as DNA and proteins can be found in Ihara et al., 1996, Nucleic Acids Res. 24: 4273-4280; Livache et al., 1995, Synthetic Metals 71: 2143-2146; Hashimoto, 1993, Supramolecular Chem. 2: 265-270; Millan et al., 1993, Anal. Chem. 65: 2317-2323.
However, most of the electrochemical tag-dependent methods known in the prior art require hybridization of the probe/target in the presence of a redox intercalator. Electrochemical detection based on redox intercalators are generally not as reproducible as redox tags that are covalently bound to an incorporated moiety. Redox intercalator methods are exceedingly dependent on washing conditions to remove excess label while not reducing the actual signal. As a consequence, false positives are often obtained using these methods. The specificity of redox intercalator methods is often much worse than can be achieved with covalently-bound redox tags.
There remains a need in this art for simple, economical, and efficient ways to detect single base extension products of nucleic acid assays for detecting mutation and genetic polymorphisms in biological samples containing a nucleic acid of interest.
This invention provides methods and apparatus for detecting mutations and genetic polymorphisms in a biological sample containing a nucleic acid of interest. Detection of single base extension using the methods and apparatus of the invention is achieved by sequence-specific incorporation of chain-terminating nucleotide species chemically labeled with an electrochemical species. In preferred embodiments, single base extension is performed using hybridization to an oligonucleotide array, most preferably an addressable array wherein the sequence of each oligonucleotide in the array is known and associated with a particular address in the array. In additional preferred embodiments, single base extension is detected using extension products labeled with electrochemical reporter groups, wherein the electrochemical reporter groups comprise a transition metal complex, most preferably containing a transition metal ion that is ruthenium, cobalt, iron or osmium.
In the practice of the methods of the invention, the invention provides an array of oligonucleotide probes immobilized to a surface that defines a first electrode. Preferably, the sequence of each oligonucleotide at each particular identified position (or xe2x80x9caddressxe2x80x9d) in the array is known and at least one of said oligonucleotides is complementary to a sequence in a nucleic acid contained in the biological sample to be assayed (termed the xe2x80x9ctargetxe2x80x9d or xe2x80x9ctarget nucleic acidxe2x80x9d). In one preferred embodiment, the sequence of at least one oligonucleotide is selected to hybridize to a position immediately adjacent to the nucleotide position in the sample nucleic acid that is to be interrogated, i.e., for mutation or genetic polymorphism. The term xe2x80x9cadjacentxe2x80x9d in this context is intended to encompass positions that are one nucleotide base upstream of base to be interrogated, i.e. in the 3xe2x80x2 direction with respect to the template strand of the target DNA. Hybridization of the oligonucleotides in the array to nucleic acid in the sample is performed in a reaction chamber and in a hybridization buffer for a time and at a temperature that permits hybridization to occur between nucleic acid in the sample and the oligonucleotides in the array complementary thereto. Single base extension is performed using a polymerase, most preferably a thermally stable polymerase, in the presence of chain-terminating primer extension units that are covalently linked to an electrochemical label. In a preferred embodiment, each chain-terminating nucleotide species (for example, di deoxy(dd)ATP, ddGTP, ddCTP and ddTTP) is labeled with a different electrochemical label, most preferably having a different, distinct and differentially-detectable reduction/oxidation potential. Single base extension is detected by applying conventional electrochemical detection methods, such as cyclic voltammetry or stipping voltammetry. Other electric or/and electrochemical methods that may also be used, include, but are not limited to, AC impedance, pulse voltammetry, square wave voltammetry, AC voltammetry (ACV), hydrodynamic modulation voltammetry, potential step method, potentiometric measurements, amperometric measurements, current step method, and combinations thereof.
In alternative embodiments, the sequence of at least one oligonucleotide is selected to hybridize to the target nucleic acid at a position whereby the 3xe2x80x2 residue of the oligonucleotide hybridizes to the nucleotide position in the sample nucleic acid that is to be interrogated for mutation or genetic polymorphism. In the array, oligonucleotides having sequence identity to the oligonucleotide that hybridizes to the target nucleic acid at it""s 3xe2x80x2 residue will also hybridize to the target, but the 3xe2x80x2 residue of such oligonucleotides will produce a xe2x80x9cmismatchxe2x80x9d with the target and will not hybridize at the 3xe2x80x2 residue. Single base extension is performed with a polymerase that will not recognize the mismatch, so that only the oligonucleotide that hybridizes to the target including at its 3xe2x80x2 residue will be extended. In these embodiments of the invention, only a single chain-terminating species labeled with an electrochemical species can be employed, or the same electrochemical species can be used for all four chain-terminating species, provided that the nucleotide sequence of each oligonucleotide in the array is known and properly associated with its position in the array. The detection of an electrochemical signal from the redox species using conventional electrochemical detection methods, such as cyclic voltammetry, at a particular position in the array thus provides the identity of the 3xe2x80x2 residue of the probe and hence the identity of the complementary nucleotide at the corresponding position in the target nucleic acid.
In the practice of a preferred embodiment of the methods and use of the apparatus of the invention, electric current is recorded as a function of sweeping voltage to the first electrode specific for each particular chain-terminating nucleotide species labeled with an electrochemically-active reporter. In preferred embodiments, current flow at each specific potential is detected at each address in the array where single base extension has occurred with the corresponding chain-terminating nucleotide species labeled with a particular electrochemical reporter group. The detection of the electrical signal at a particular position in the array wherein the nucleotide sequence of the oligonucleotide occupying that position is known enables the identity of the extended nucleotide, and therefore the mutation or genetic polymorphism, to be determined.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.